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The Isolation and Genetic Characterisation of a Novel Alphabaculovirus for the Microbial Control of Cryptophlebia Peltastica and Closely Related Tortricid Pests

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The Isolation and Genetic Characterisation of a Novel Alphabaculovirus for the Microbial Control of Cryptophlebia Peltastica and Closely Related Tortricid Pests RHODES UNIVERSITY Where leaders learn The isolation and genetic characterisation of a novel alphabaculovirus for the microbial control of Cryptophlebia peltastica and closely related tortricid pests Submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY At RHODES UNIVERSITY By TAMRYN MARSBERG December 2016 ABSTRACT Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) is an economically damaging pest of litchis and macadamias in South Africa. Cryptophlebia peltastica causes both pre- and post-harvest damage to litchis, reducing overall yields and thus classifying the pest as a phytosanitary risk. Various control methods have been implemented against C. peltastica in an integrated pest management programme. These control methods include chemical control, cultural control and biological control. However, these methods have not yet provided satisfactory control as of yet. As a result, an alternative control option needs to be identified and implemented into the IPM programme. An alternative method of control that has proved successful in other agricultural sectors and not yet implemented in the control of C. peltastica is that of microbial control, specifically the use of baculovirus biopesticides. This study aimed to isolate and characterise a novel baculovirus from a laboratory culture of C. peltastica that could be used as a commercially available baculovirus biopesticide. In order to isolate a baculovirus a laboratory culture of C. peltastica was successfully established at Rhodes University, Grahamstown, South Africa. This is the first time a laboratory culture of C. peltastica has been established. This allowed for various biological aspects of the pest to be determined, which included: length of the life cycle, fecundity and time to oviposition, egg and larval development and percentage hatch. The results obtained from these studies found that the biology of C. peltastica was similar to that of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). Once the laboratory culture had reached high densities, larvae showing symptoms of baculovirus infection were observed. Symptomatic larvae were collected and examined for the presence of a baculovirus. An alphabaculovirus (NPV) was successfully isolated and morphologically identified using purified OBs that were sectioned and observed by transmission electron microscopy. This was then confirmed by amplifying the polyhedrin gene region using degenerate primers. A BLAST analysis found a 93% similarity to a partial polyhedrin gene sequence to be that of Epinotia granitalis (Butler) (Lepidoptera: Tortricidae). The alphabaculovirus was then genetically characterised by generating restriction profiles and sequencing the whole genome. Due to the novelty of the virus, no comparison could be made. i The biological activity of the alphabaculovirus was then tested against C. peltastica and two closely related Tortricidae pests: T. leucotreta and Cydiapomonella (Linnaeous) (Lepidoptera: Tortricidae). The alphabaculovirus was highly virulent against all three species. The lethal concentrations (LC50 and LC90) for the virus against C. peltastica was 8.19 x 103 and 3.33 x 105 OBs/ml. The LC50 and LC90 for T. leucotreta was 2.29 x 103 and 9.97 x 104 OBs/ml, respectively and C. pomonella had a LC50 of 1.43 x 103 OBs/ml and LC90 1.26 x 104 OBs/ml. The virus was particularly virulent against T. leucotreta and C. pomonella as compared to C. peltastica. The biological activity of the alphabaculovirus was also tested against CpGV resistant European C. pomonella. From the results it was observed that the virus had the ability to overcome the resistance in C. pomonella and could potentially be used in the resistance management of C. pomonella. With the successful biological activity results obtained from this study, preliminary investigation were made into the mass production of the alphabaculovirus using both the in vivo and in vitro production methods. For in vivo production both the homologous host (C. peltastica) and a heterologous host (T leucotreta) were investigated. Preliminary studies focused on determining the biological activity in fifth instars of both hosts. Fifth instar LC50 and LC90 values for C. peltastica were 3.43 x 103 and 1.11 x 107 OBs/ml and for T. leucotreta the LC50 and LC90 values were 2.53 x 1 0 3 and 8.82 x 1 0 6 OBs/ml, respectively. The average yield of virus produced in each species was also determined. Cryptophlebia peltastica had the highest viral yield of 5.37 x 1010 OBs/larva and 2.93 x 1010 OBs/larva for T. leucotreta. The results obtained, from the preliminary investigation concluded that the virus could be produced in vivo in both C. peltastica and T. leucotreta, however further research is required into the mass production in both hosts. The in vitro production of the virus was also considered and the susceptibility of the virus was tested against the C. pomonella cell line, Cp14R. After infection of the Cp14R cells with budded virus collected from fifth instar C. peltastica larvae, OBs could be observed after three days. Thus, the alphabaculovirus is susceptible to the Cp14R cell line, thus has the potential to be produced in vitro and further characterised. This study is the first to report of the identification and characterisation of a novel alphabaculovirus isolated from a laboratory reared culture of C. peltastica and the potential for it to be commercially developed into a bipoesticide and used against Tortricidae pests. ii TABLE OF CONTENTS ABSTRACT.......................................................................................................................................i TABLE OF CONTENTS.............................................................................................................. iii LIST OF TA BLES........................................................................................................................... x LIST OF FIG URES......................................................................................................................xiv ABBREVIATIONS....................................................................................................................... xxi ACKNOWLEDGEMENTS............................................................. xxv CHAPTER 1: GENERAL INTRODUCTION......................................................................... 1 1.1 Introduction................................................................................................................................. 1 1.1.1 The host plant................................................................................................................1 1.1.2 The pest: Cryptophlebia peltastica............................................................................6 1.1.2.1 Pest Description.......................................................................................... 7 1.1.2.2 Life C ycle......................................................................................................7 1.1.2.3 Damage and control techniques of Cryptophlebia peltastica................8 1.1.3 Microbial Control..................................................................................................... 10 1.1.4 Baculoviruses........................................................................................................... 13 1.1.4.1 Identification and characterisation of a baculovirus.............................. 17 1.1.4.2 Biological activity.................................................................................... 19 1.1.4.3 Mass production....................................................................................... 19 1.2 Research A im s.............................................................................................................................20 CHAPTER 2: LABORATORY REARING AND STUDY OF THE BIOLOGY OF CRYPTOPHLEBIA PELTASTICA....................................................................21 iii 2.1 Introduction.................................................................................................................................21 2.2 Materials and M ethods.............................................................................................................. 23 2.2.1 Culture rearing............................................................................................................ 23 2.2.2 Fecundity and time to oviposition............................................................................25 2.2.3 Egg development........................................................................................................25 2.2.4 Percentage hatch.........................................................................................................26 2.2.5 Larval development....................................................................................................26 2.2.6 Pupal development.................................................................................................... 27 2.3 Results..........................................................................................................................................27 2.3.1 Culture rearing, sex ratio and difference between male and female pupae and adults..................................................................................................................................... 27 2.3.2 Fecundity and time to oviposition............................................................................29 2.3.3
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